Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

Oligomerization domains in the glycan-binding receptors DC-SIGN and DC-SIGNR: Sequence variation and stability differences.

Human dendritic cell-specific intercellular adhesion molecule-1 grabbing nonintegrin, DC-SIGN, and the sinusoidal endothelial cell receptor DC-SIGNR or L-SIGN, are closely related sugar-binding receptors. DC-SIGN acts both as a pathogen-binding endocytic receptor and as a cell adhesion molecule, while DC-SIGNR has only the pathogen-binding function. In addition to differences in the sugar-binding properties of the carbohydrate-recognition domains in the two receptors, there are sequence differences in the adjacent neck domains, which are coiled-coil tetramerization domains comprised largely of 23-amino acid repeat units. A series of model polypeptides consisting of uniform repeat units have been characterized by gel filtration, differential scanning calorimetry and circular dichroism. The results demonstrate that two features characterize repeat units which form more stable tetramers: a leucine reside in the first position of the heptad pattern of hydrophobic residues that pack on the inside of the coiled coil and an arginine residue on the surface of the coiled coil that forms a salt bridge with a glutamic acid residue in the same polypeptide chain. In DC-SIGNR from all primates, very stable repeat units predominate, so the carbohydrate-recognition domains must be held relatively closely together. In contrast, stable repeat units are found only near the membrane in DC-SIGN. The presence of residues that disrupt tetramer formation in repeat units near the carbohydrate-recognition domains of DC-SIGN would allow these domains to splay further apart. Thus, the neck domains of DC-SIGN and DC-SIGNR can contribute to the different functions of these receptors by presenting the sugar-binding sites in different contexts.

Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A.

The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl- channels. Members of both these branches are activated by Ca2+, acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca2+-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca2+-activated Cl- channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca2+ Constructs combining subunits with different potencies for Ca2+ show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca2+ binding to sites that are embedded within the transmembrane part of each subunit.

X-ray structure of a calcium-activated TMEM16 lipid scramblase.

The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.